Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 28, 2016; 22(16): 4109-4119
Published online Apr 28, 2016. doi: 10.3748/wjg.v22.i16.4109
Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines
Gursimran Chandhok, Judit Horvath, Annu Aggarwal, Mohit Bhatt, Andree Zibert, Hartmut HJ Schmidt
Gursimran Chandhok, Andree Zibert, Hartmut HJ Schmidt, Klinik für Transplantationsmedizin, Universitätsklinikum Münster, D-48149 Münster, Germany
Judit Horvath, Institut für Humangenetik, Westfälische Wilhelms-Universität, D-48149 Münster, Germany
Annu Aggarwal, Mohit Bhatt, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai 400103, India
Author contributions: Chandhok G, Aggarwal A, Bhatt M, Zibert A and Schmidt HHJ designed the experiments; Chandhok G and Horvath J performed the experiments; Chandhok G interpreted the data and performed statistical analysis; Chandhok G, Zibert A and Schmidt HHJ wrote the manuscript; all authors read and approved the final manuscript.
Conflict-of-interest statement: The authors declare no conflict of interest associated with this manuscript.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Hartmut HJ Schmidt, MD, Professor, Klinik für Transplantationsmedizin, Universitätsklinikum Münster, Albert-Schweitzer-Campus 1, Gebäude A14, D-48149 Münster, Germany. hepar@ukmuenster.de
Telephone: +49-251-8357935 Fax: +49-251-8357771
Received: December 5, 2015
Peer-review started: December 7, 2015
First decision: January 13, 2016
Revised: January 24, 2016
Accepted: February 20, 2016
Article in press: February 22, 2016
Published online: April 28, 2016
Processing time: 136 Days and 9.5 Hours
Abstract

AIM: To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture.

METHODS: The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined.

RESULTS: Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability.

CONCLUSION: The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines.

Keywords: ATP7B; D-penicillamine; Zinc; Mutations; Wilson disease; Therapy

Core tip: Copper overload in Wilson disease (WD) is treated with anti-copper therapy. However, the effect of treatment has not been studied using human hepatic cells lacking the ATP7B copper transporter. Using a previously established ATP7B KO cell line, we generated stable mutant cell lines to study functional analysis and response to zinc (Zn) and D-penicillamine (DPA). All mutants showed individual response rates following copper and anti-copper treatment. Highest rescue from copper toxicity was observed after combined Zn and DPA treatment. The study provides novel insights into genotype-phenotype correlations and genotype-specific treatment of WD.